Lymph node (LN) is a secondary lymphoid organ with highly organized and compartmentalized structure. LNs harbor B, T, and other cells among fibroblastic reticular cells (FRCs). FRCs are characterized by both podoplanin (PDPN/gp38) expression and by the lack of CD31 expression. FRCs are involved in several immune response processes but mechanisms underlying their function are still under investigation. Double-negative cells (DNCs), another cell population within LNs, are even less understood. They do not express PDPN or CD31, their localization within the LN is unknown, and their phenotype and function remain to be elucidated. This study evaluates the gene expression and cytokines and chemokines profile of human LN-derived FRCs and DNCs during homeostasis and following inflammatory stimuli. Cytokines and chemokines secreted by human FRCs and DNCs partially diverged from those identified in murine models that used similar stimulation. Cytokine and chemokine secretion and their receptors expression levels differed between stimulated DNCs and FRCs, with FRCs expressing a broader range of chemokines. Additionally, dendritic cells demonstrated increased migration toward FRCs, possibly due to chemokine-induced chemotaxis since migration was significantly decreased upon neutralization of secreted CCL2 and CCL20. Our study contributes to the understanding of the biology and functions of FRCs and DNCs and, accordingly, of the mechanisms involving them in immune cells activation and migration.

Lymph node is a secondary lymphoid organ with highly organized and compartmentalized structure. Fibroblastic reticular cells (FRC) integrate LN structure, and are characterized by podoplanin expression (PDPN) and lack of CD31. FRCs are involved in several immune response processes but the mechanism is still under investigation. Another cell population present in the lymph node is the one lacking PDPN and CD31, so called, double negative population (DNC). FRCs and DNCs are described as chemokines producers, controlling migratory pattern and positioning of immune cells during homeostasis or inflammation. We evaluate the gene expression of human FRCs and DNCs after an inflammatory stimulus. Lymph nodes were obtained from 04 patients with different clinical diagnosis submitted to surgical procedures. All patients signed an informed consent (Approved by Ethic Committee - CAAE: 07768712.4.0000.0071). All lymph nodes were analyzed by histopathology, they were also enzymatic digested. Cells were characterized and sorted in two populations FRCs and DNCs. These cells were maintained without stimuli or stimulated with TNFα + IL-1β for 24hours. Next, RNA was extracted; gene expression was characterized using DNA Microarray. Our results demonstrate up-regulation of chemokines such as CCL2, CCL3, CCL5, CCL7 CXCL1, CXCL2, CXCL8 and CXL10 (p<0.05) in both cells subsets after inflammatory stimuli, the secretion enhancement of CCL2, CCL20 and CXCL8 was confirmed by ELISA. The up-regulated expression of CCL3L3, CCL11, CCL13, CXCL5, CXCL6 only by FRCs and CCL8, CXCL3 only by DNCs, may link these cells to additional properties that need to be further investigated. These results show that FRC and DNC positively respond to an inflammatory stimulus.

Lymph node (LN) is a secondary lymphoid organ with highly organized structure. Fibroblastic reticular cells (FRCs) integrate LN structure, and are characterized by podoplanin expression (PDPN, gp38) and lack of CD31. FRCs are involved in several immune response processes but the mechanism is still under investigation. Another cell population present in LN is a fibroblastic cell lacking PDPN and CD31 expression, called double negative cells (DNCs). FRCs and DNCs are described to control migratory pattern and positioning of immune cells during homeostasis or inflammatory response. We evaluate in human derived FRCs and DNCs the gene expression after IFN-γ-treatment and found up-regulated genes related to antigen processing and MHC class I and II up-regulated. MHC class I is expressed by all nucleated cells and MHC class II constitutive expression is confined to the immune system antigen-presenting cells, but can be induced in several cells types. MHC class II genes control has been described to play a role in eliminting foreign antigens, while minimizing autoreactive responses against self-antigens. MHC class II molecules can fulfill additional functions by triggering a variety of signaling pathways, they can regulate proliferation and maturation to apoptosis. In addition, we were able to demonstrate that HLA-ABC and HLA-DR were increased in the membrane of both FRCs/DNCs derived from different patients/lymph nodes after treatment with IFN-γ. FRCs seem to be more responsive to IFN-γ than DNCs. Furthermore, we have tested the ability of FRCs to internalize particles, showing a functional MHC II pathway.